Pharmacological inhibition of HERG (human ether-a-go-go-related gene) potassium channels by therapeutically diverse drugs is considered to involve a binding site within the inner cavity, involving aromatic amino-acids in the channel inner (S6) helix which are rendered accessible on channel gating (Mitcheson & Perry, 2003). However, the antimycotic drug ketoconazole has been suggested previously to inhibit HERG channel current (I_HERG) without a requirement for channel activation, based on observations made using the Xenopus oocyte expression system (Dumaine et al. 1998). The aim of the present study was to determine whether or not ketoconazole inhibits I_HERG from a mammalian (Human Embryonic Kidney, HEK293) cell line, independently of channel gating. Whole-cell patch-clamp measurements of I_HERG were made at 37°C using HERG stably expressed in HEK293 cells. Five different concentrations of ketoconazole ranging from 50 nM to 50 μM were applied (n = at least 5 cells per concentration) and inhibition of I_HERG tails at -40 mV (following a 2 s activating pulse from -80 mV to +20 mV) was monitored. Ketoconazole inhibited I_HERG tails with a half-maximal inhibitory concentration (IC₅₀) of 1.8 μM (C.I. 965.0 nM to 3.4 μM). The profile of I_HERG blockade during a 12 s ascending voltage ramp between -80 and +40 mV (n = 5 cells) was inconsistent with a predominantly closed-state channel blocking effect and blockade was also observed to be time-dependent during application of a sustained depolarisation from -80 to 0 mV (n = 6 cells). Importantly, the S6 mutant F656A (phenylalanine to alanine) significantly attenuated the inhibitory effect of ketoconazole (20 μM ketoconazole inhibited WT-HERG by 88.6 ± 1.7%; mean ± S.E.M.; n = 5 and F656A-HERG by 24.9 ± 3.4 %; n = 5 p < 0.001; unpaired Student's t test). These observations provide evidence that ketoconazole inhibits I_HERG by gaining access to its binding site on channel gating; thus is inconsistent with a major role for closed-state channel inhibition by this agent.